The present invention relates generally to methods and compositions of erythroid cells generated from human embryonic stem cells (hESCs). More particularly, the present invention relates to methods and compositions of erythroid cells that are at least CD31−, CD34−, CD71+ and CD235a+, and express adult β-hemoglobin and fetal γ-hemoglobin, but not embryonic ζ-hemoglobin.
Hematopoiesis is a formation of blood cell components from stem cells, typically hematopoietic stem cells. Prenatally, hematopoiesis occurs in the yolk sack, then the liver, and eventually the bone marrow. In normal adults, however, it occurs in bone marrow and lymphatic tissues. It has been estimated that there is approximately 1 hematopoietic stem cell per 104 bone marrow cells.
The blood cells produced during hematopoiesis are divided into the following three cell lineages: (1) erythroid cells, (2) lymphoid cells, and (3) myeloid cells. Erythroid cells, including normoblasts, erythroblasts and mature red blood cells (RBCs), are the most common type of blood cell and are a principal means of delivering oxygen from the lungs to body tissues. Lymphoid cells, including B-cells and T-cells, are a type of white blood cell that play a significant role in the body's immune defenses. Myeloid cells, including granulocytes, megakaryocytes, and macrophages, are a diverse group of cells comprising other white blood cells (e.g., neutrophils, eosinophils and basophils) and platelets. Of particular interest herein is the generation of cells of the erythroid lineage.
“Erythropoiesis” is a formation of erythroid cells from stem cells, typically from hematopoietic stem cells. In an average adult, production of mature RBCs (erythrocytes) equals their loss. As such, the average adult produces 3.7×1011 RBCs/day.
Given the paucity of hematopoietic stem cells, researchers have recently shifted their attention to generating RBCs from embryonic stem cells (ESCs), especially hESCs. hESCs offer an opportunity to generate RBCs in sufficient quantities to study the differentiation of RBCs in vitro. More importantly, RBCs generated from hESCs would provide a safe and an ample alternative source of cells for transfusion, as well as for treating conditions involving defective RBCs (e.g., hypoxia and sickle cell anemia). In the United States, for example, only five percent of eligible donors across the nation donate blood; however, the number of transfusions nationwide increases by nine percent every year.
Recently, Umeda et al. showed that primate ESCs co-cultured with OP9 stromal cells generated cells that expressed embryonic, fetal and adult hemoglobin. Umeda K, et al., “Sequential analysis of alpha- and beta-globin gene expression during erythropoietic differentiation from primate embryonic stem cells,” Stem Cells 24:2627-2636 (2006). However, Umeda et al. cultured the cells in serum, which may be problematic due to the uncharacterized composition and variation of serum. Moreover, erythroid cells generated by Umeda et al.'s method contained 5% to 15% myeloid cells.
Likewise, Olivier et al. showed that hESCs co-cultured with human fetal liver cells generated CD34+ cells that produced embryonic and fetal hemoglobin. Olivier E, et al., “Large-scale production of embryonic red blood cells from human embryonic stem cells,” Exp. Hematol. 34:1635-1642 (2006); for similar results, see also Qiu C., et al., “Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver cells recapitulates the globin switch that occurs early in development,” Exp. Hematol. 33:1450-1458 (2005). Unfortunately, Olivier et al.'s cells did not produce adult hemoglobin and retained expression of embryonic ζ-hemoglobin.
Other researchers have also generated RBCs from ESCs; however, these methods either used non-human/non-primate stem cells or used an embryoid body-dependent method (i.e. no direct differentiation). These methods, however, produced a mixture of erythroid and myeloid cells. See Carotta S, et al., “Directed differentiation and mass cultivation of pure erythroid progenitors from mouse embryonic stem cells,” Blood 104:1873-1880 (2004); Chadwick K, et al., “Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells,” Blood 102:906-915 (2003); Kaufman D, et al., “Hematopoietic colony-forming cells derived from human embryonic stem cells,” Proc. Natl. Acad. Sci. USA 98:10716-10721 (2001); Ng, E, et al., “Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation,” Blood 106:1601-1603 (2005); and Zambidis E, et al., “Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development,” Blood 106:860-870 (2005).
For the foregoing reasons, there is a continuing need for alternative methods of generating erythroid cells from hESCs, especially erythroid cells that express adult hemoglobin, that are generated under plasma/serum-free conditions and that are free of lymphocytes.